Project 4, headed by Dr. Theresa Pizarro, will focus on epithelial-immune cell interactions, specifically the interplay between the intestinal epithelium, DCs and T cells. The central hypothesis is that intestinal DCs prematurely exposed to luminal antigens as a direct consequence of an inherent defect in intestinal epithelial barrier function leads to aberrant innate DC activation that drives dysregulated adaptive immunoregulatory processes, resulting in uncontrolled chronic gut inflammation similar to that observed in CD. Specific Aim 1 will characterize epithelial-DC interactions leading to activation of the intestinal lamina propria (LP) DC population. DCs will be purified from the LP and mesenteric lymph nodes (MLNs) of SAMP, SAMP-ChrX*B6, as well as control AKR and 86 mice. Epithelial-DC interactions will be investigated by a variety of techniques, including cytokine and chemokine measurement, FACS and in vitro cell culture analysis and static confocal and dynamic 2-photon microscopy. Finally, the impact of the bacterial flora on epithelial-DC interactions will be evaluated in germ-free (GF), compared to specific pathogen-free (SPF) SAMP mice. In Aim 2, we will determine how inherent epithelial barrier dysfunction results in loss of intestinal resident DC anergy/tolerance and consequently drives dysregulated T regulatory cell (Treg) function. The ability of DCs from the SAMP, SAMP-ChrX*B6, and controls to polarize Teff/Treg cell populations will be assessed by multiple techniques, both in vitro and in vivo to precisely define DC/Treg interaction in this model. Aim 3 will evaluate the precise genetic contribution of ChrX-linked genes to the development of chronic ileitis in the SAMP strain. B6-ChrX*SAMP will be generated and fine gene mapping will be performed to identify and potentially confirm candidate disease susceptibility genes on ChrX (i.e., claudin-2, TLR cluster, FoxP3) previously shown to be dysregulated in SAMP mice. The overall goal of this project is to further elucidate the mechanism(s) of chronic intestinal inflammation characteristic of CD, and identify potential, early disease pathways that can lead to targeted therapeutic strategies to treat patients suffering from this devastating disease.